The overall goals of this project will be to test potential inhibitors and modulators of polyamine content, to aid in the design of such inhibitors, to validate the physiological relevance of the structures of AdoMetDC, SSAT and aminopropyltransferases and to provide additional proteins that will yield informative structures. All of the techniques needed for the proposed studies are currently available and a large number of relevant mutant proteins of human AdoMetDC and SSAT have already been made by site-directed mutagenesis. The project has 7 inter-related specific aims. Aims 1-5 will be started immediately. Preliminary studies using compounds currently available will be used to set up and validate the experimental protocols for aims 6 and 7 and these aims will be phased in as promising new compounds and structures become available. Routine techniques are available for the production of further mutants by site-directed mutagenesis and for the purification of the needed proteins. The detailed specific aims are: (1) derivation and characterization of wild type and mutant forms of AdoMetDC; (2) studies of inhibitors of AdoMetDC activity including examination of new compounds and studies of the interaction of inhibitors with key residues in the protein; (3) investigation of the inhibition of the processing of AdoMetDC in vitro; (4) investigation of the binding of putrescine that brings about activation of AdoMetDC; (5) investigation of the interaction of polyamine analogs with SSAT leading to changes in structure and stabilization; (6) studies of key residues in aminopropyltransferase mediating interaction with substrates and possible inhibitors; (7) investigation of the effects of potential inhibitors of AdoMetDC and inducers of SSAT on polyamine content and cell growth in vitro using human tumor cells. The biochemical studies that will be carried out in this project will validate the relevance of structures determined in Program 2; provide proteins for structural determination in Program 2 that are most likely to be informative; evaluate models produced in Program 3; and test potential inhibitors and modulators produced in Program 4.